Team:Heidelberg/Notebook/Sensing Group/Notebook/5thweek

Revision as of 15:46, 26 October 2008 by Koljaschleich (Talk | contribs)

Monday, 09/01/2008

1.selection for Fusion-1 and Fusion-2 cloning
the DNA of the colonies was extracted with MINIprep, and then screen with test digestion: PstI (Buffer3+BSA at 37)
- for Fusion-1: if our insert is there: one band of 6101 bp if no inser: two bands: 4388 + 2213 bp
- for Fusion-2: if our insert is there: one band of 6122 bp if no inser: two bands: 4388 + 2213 bp

PCR of LuxQ and two fusion protein constructs

Lyse V. harveyi for 5 min @ 99 °C
PCR with Phusion (template: supernatend of boiled V. harveyi (a), colony (b))
- 5 min @ 98 °C || 20s @ 98 °C | 40s @ 58 °C | 1min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
- 35(34) µl H₂O, 10 µl 5x HF, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Phusion, 0(1) µl DMSO
PCR with Pfu
- 3 min @ 94 °C || 45s @ 94 °C | 45s @ 58 °C | 2min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
- 40(39) µl H₂O, 5 µl 10x Buffer, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Pfu, 0(1) µl DMSO